This MIRA grant proposal briefly summarizes the activities currently supported by two NIGMS grants and my vision for the evolution of this research over the next five years. The common theme that unifies this research is a unique combination of the development and application of new physical methods that can be broadly applied to the quantitative analysis of biological systems. The long-term goals of GM27738 (Electrostatics and Dynamics in Proteins) are to develop spectroscopic methods for probing electric fields in proteins and to apply these methods to obtain quantitative information on fields and their effects on function at the active sites of several enzymes. We have led the development of vibrational Stark spectroscopy as a general approach to map these fields. Recent and continuing work emphasizes the connection between electrostatics and catalysis where we can, for the first time, estimate the electrostatic contribution to the catalytic proficiecy of enzymes. We also study dynamics in green fluorescent protein (GFP), which we began to study reasoning that the native chromophore could be used to probe solvation dynamics (time dependent electric fields) in proteins. Instead we discovered that GFP exhibits excited state proton transfer. Recent work emphasizes novel applications of split GFP where light-driven association and dissociation reactions have been discovered. We seek to understand the basic mechanism(s) of these processes and optimize them for optogenetic and imaging applications. The long-term goals of GM069630 (Membrane Fusion and Dynamics Using Supported Bilayers) are to develop methods to probe the organization and dynamic reorganization of lipids and proteins in biological membranes and to apply these methods to problems of broad biological importance. Our lab has pioneered the development of model membrane architectures, along with imaging and analytical methods, that probe fundamental aspects of membrane organization and dynamics. We use these approaches to address open questions in three areas of current biological significance. (i) The mechanism of vesicle and enveloped viral membrane fusion is studied using model membrane architectures we developed as targets to precisely probe the steps of fusion at the single event level. (ii) The organization of lipids and membrane proteins are characterized with unprecedented lateral resolution using imaging mass spectrometry, emphasizing the correlated motion of lipid and protein components believed to be associated in rafts. (iii) A novel membrane interferometer has been designed with the ultimate goal of correlated measurements of integral membrane protein conformational changes by optical interferometry and electrical activity by electrophysiological methods. Each of these areas represents a major current challenge in membrane biophysics and biology.